Apparatus for generating electrical pulses and methods of using same

ABSTRACT

A method and apparatus are provided for delivering an agent into a cell through the application of nanosecond pulse electric fields (“nsPEF&#39;s”). The method includes circuitry for delivery of an agent into a cell via known methods followed by the application of nanosecond pulse electric fields to said cell in order to facilitate entry of the agent into the nucleus of the cell. In a preferred embodiment, the present invention is directed to a method of enhancing gene expression in a cell comprising the application of nanosecond pulse electric fields to said cell. An apparatus for generating long and short pulses according to the present invention is also provided. The apparatus includes a pulse generator capable of producing a first pulse having a long duration and low voltage amplitude and a second pulse having a short duration and high voltage amplitude.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. patent applicationSer. No. 10/564,994, filed Jul. 24, 2006, which is a 371 application ofPCT/US2004/023078, filed Jul. 19, 2004, which claims the benefit of U.S.Provisional Application No. 60/487,932, filed Jul. 18, 2003, U.S.Provisional Application No. 60/499,921, filed Sep. 4, 2003, and U.S.Provisional Application No. 60/526,585, filed Dec. 4, 2003, thedisclosures of which are incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

The present invention was made with Government support under AFOSR MURIGrant No. F49620-02-1-0320 awarded by the United States Air Force Officeof Scientific Research. The Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Both the detection and use of electricity/electric fields in medicineand biology are widespread and well accepted. Electrocardiography (EKG)and electroencephalography (EEG) are used to detect electrical activityin the heart and brain, respectively. Cardioversion, the application ofa pulsed electric field to heart muscle, is routinely used to stop,modify, or re-start the heart's beating. Low power electric fields canbe applied to bone fractures to stimulate healing. Electromyography, theapplication of measured electrical pulses to muscles or their associatednerves, can be used to measure muscle function and/or judge the degreesof muscle damage. In biology, electric fields have various applicationsand can be used, for example, to separate molecules of different sizes(electrophoresis) or different charges (isoelectric focusing), and toseparate cells with different characteristics (cell sorting during flowcytometry). Electric fields can also be used to facilitate entry of newproteins or genes into living cells via a process calledelectroporation.

In electroporation, application of brief (on the scale ofthousandths-to-millionths of a second), moderate power (kilovolt/meter)electric fields causes permeabilization (leakiness) of the cell'ssurface membrane which then allows entry of materials/molecules into thecell that would otherwise never gain access to the cell's interior.After the initial permeabilization of the cell membrane, the celleventually returns to its normal “non-leaky” condition. Now, however,the cell will carry and/or utilize the materials that have beenintroduced into it by the electroporation. This process can be used forthe introduction of genes or drugs into a cell, for example, fortransdermal drug delivery (Neumann, E., Kakorin, S., and Toensing, K.(1999), Fundamentals of electroporative delivery of drugs and genes.Bioelectrochem. Bioenerg. 48, 3-16.1999; Weaver, J. C., Vaughan, T. E.,and Chizmadzhev, Y. (1999), Theory of electrical creation of aqueouspathways across skin transport barriers. Adv. Drug Deliv. Rev. 35,21-39), and as a therapeutic tool for the treatment of cancer usingelectrochemotherepy (Belehradek, M., Domenge, C., Luboinski, B.,Orlowski, S., Belehradek, J. Jr., and Mir, L. M. (1993),Electrochemotherapy, a new antitumor treatment. First clinical phaseI-II trial. Cancer 72, 3694-700.1993; Heller, R., Jaroszeski, M. J.,Glass, L. F., Messina, J. L., Rapaport, D. P., DeConti, R. C., Fenske,N. A., Gilbert, R. A., Mir, L. M., Reintgen, D. S. (1996), Phase I/IItrial for the treatment of cutaneous and subcutaneous tumors usingelectrochemotherapy. Cancer 77, 964-71.1996; Hofmann, F., Ohnimus, H.,Scheller, C., Strupp, W., Zimmermann, U., and Jassoy, C. (1999),Electric field pulses can induce apoptosis. J. Membr. Biol. 169,103-109). Electrochemotherapy or electroporation therapy (EPT) is amethod for the in vivo delivery of poorly permeable chemotherapeuticagents, such as bleomycin, to tumor cells that can be appropriatelyoriented between two electrodes (Dev S. B., Hofmann, G. A.,Electrochemotherapy—a novel method of cancer treatment. Cancer Treat Rev20:105-15, 1994; Hofmann et al., Electroporation therapy: a new approachfor the treatment of head and neck cancer. IEE Trans Biomed Eng46:752-9, 1999; Mir, L. M., Orlowski, S. Mechanisms ofelectrochemotherapy. Adv Drug Deliv Rev., 35:107-118, 1999). Bothelectroporation and EPT are dependent on electric effects on the plasmamembrane of the cells or tissues.

Electroporation occurs with pulse durations on the order of 0.1 to 20milliseconds (“ms”) (Dev, S. B., Rabussay, D. A., Widera, G., andHofmann, G. A. (2000) IEEE Trans. Plasma Sci. 28, 206-223) with electricfields on the order of volts to low kilovolts/centimeter; however,specific conditions depend on the particular cell type and the cellsuspension media. These millisecond pulses promote transient membraneporation and cell survival. Alternatively, using different electrical orcellular conditions, electroporation can cause rupture/death of cells.Although the physical nature of the pores is not well characterized, theexperimental conditions that allow intracellular delivery of membraneimpermeable molecules with good cell survivability are well known.Conditions for optimal electroporation depend on the waveform, theconstituents of the media in which the cell is suspended, and the celltype (Weaver, J. C., Electroporation of cells and tissues, in: J. D.Bronzino (Ed.), The Biomedical Engineering Handbook, CRC and IEEE press,Boca Raton, Fla., 1995, pp. 1431-1440; Djuzenova et al., Effect ofmedium conductivity and composition on the uptake of propidium iodideinto electropermeabilized myeloma cells. Biochim Biophys Acta,1284:143-52, 1996). In any case, the electroporation effects of thesemillisecond low power applied electric fields occur only at the cell'ssurface membrane.

As mentioned above, electric fields and the process of electroporationhave also been used for the introduction of genes into cells. Thetransfection of living cells with DNA is a common molecular techniqueused to express exogenous genes in cells for transcription studies orfor therapeutic purposes in the treatment of some diseases. Knowntransfection methods include the incorporation of DNA into lipidvesicles for fusion with the plasma membrane, the endocytosis of DNAprecipitated with calcium phosphate or dextran, the use of viral vectorsthat infect the cell with the gene of interest, andelectropermeabilization or electroporation using pulsed electric fieldsthat form “pores” in the plasma membrane. Some cell types, especiallythose that grow in suspension, can only be effectively transfected byelectropermeabilization. Enhanced or optimized gene expression has beenpreviously accomplished using classical electroporation pulses bychanging the pulse duration of a long pulsed electric field (forexample, within the range of 1 microsecond-20 milliseconds), changingthe electric field intensity within classical electroporation range(0.1-5 kV/cm), and/or by modifying the conductivity of the buffer ormedia. In other transfection procedures enhanced gene expression hasbeen accomplished by changing the concentration of DNA used in thetransfection procedure, changing the physical/chemical properties duringtransfection (pH, ionic strength, etc), using various lipid combinationswith different properties, or adding other constituents to the cellculture media or buffers to aid transfection efficiency.

Even with these known techniques, more efficient methods of introducingan agent into a cell and new methods of enhancing gene expression arestill needed. These and various other needs are addressed, at least inpart, by one or more embodiments of the present invention.

SUMMARY OF THE INVENTION

The present invention is directed to a method of introducing an agentinto a cell comprising the application of nanosecond pulse electricfields (“nsPEF's”).

In accordance with one or more embodiments of the invention, a methodfor introducing an agent into a cell includes providing a preparationcomprising the cell and agent, and applying the nanosecond pulseelectric fields to said preparation, which facilitates the entry of theagent into the nucleus. The nsPEFs can range in time from 1 to 1000nanoseconds, preferably 1 to 300 nanoseconds. The nsPEFs can also rangein electric field intensity from 1 to 1000 kV/cm, preferably 10 to 350kV/cm. The agent may be selected from the group comprising drugs,nucleic acids, protein, peptides, and polypeptides, for example.

Various embodiments of the invention allow the drug to be an antibioticor a chemotherapeutic agent selected from the group comprisingbleomycin, daunomycin, 5-FU, cytosine arabinoside, colchicine,cytochalasin B, daunorubicin, neocarcinostatin, suramin, doxorubicin,carboplatin, taxol, mitomycin C, vincristine, vinblastine, methotrexate,and cisplatin, and suitable combinations thereof. Furthermore, the agentcan be a nucleic acid, wherein the nucleic acid is selected from thegroup comprising DNA, cDNA, and RNA. According to the present invention,these nucleic acids may encode a homologous or heterologous gene productand the cell can be transfected so that this gene product is expressedin the cell. The nucleic acid can also be an expression vector whereinthe expression vector contains a homologous or heterologous nucleic acidencoding a gene product operably linked to a suitable promoter sequence.The nucleic acid may also modify the expression of a gene and providegene therapy, for example. The nucleic acid introduced into the cell mayalso modulate cell proliferation or elicit an immune response. Furtherembodiments of the invention provide for agents that can be in the forma polypeptide, wherein the polypeptide is selected from the groupcomprising a hormone, a cytokine, a lymphokine, a growth factor, or acombination thereof. The polypeptide can also be antigen or an antibody.

In accordance with additional embodiments of the invention, the agentcan be a cytotoxic agent selected from the group comprising ricin,abrin, diphtheria toxin, and saporin. Any type of cell may be used inthe present invention including eukaryotic cells, prokaryotic cells, fatcells, bone cells, vascular cells, muscle cells, cartilage cells, stemcells, hematopoeitic cells, lung cells, airway cells, liver cells,intestinal cells, skin cells, nerve cells, cancer cells, bacterialcells, and combinations thereof.

In accordance with at least one embodiment of the invention, a method ofenhancing gene expression includes providing a preparation comprisingthe cell and the nucleotide sequence to be delivered into the cell, andapplying nanosecond pulse electric fields to said preparation, whereinsaid application facilitates the entry of the agent into the nucleus.

In other forms of the invention, a method of enhancing gene expressionin a cell includes transfecting a cell with a desired gene and applyingnanosecond pulse electric fields to a cell. The cell may be transfectedby any commonly known method, including but not limited to,electroporation, the use of lipid vesicles, the use of viral vectors,and/or co-precipitation with calcium phosphate or dextran.

In various embodiments of the invention, a method of enhancing geneexpression in a cell includes applying one or more long pulses to a celland applying one or more nanosecond pulse electric field pulses to saidcell. These long pulses can range in duration from about 0.001 to 30milliseconds, preferably about 0.1 to 20 milliseconds, and can haveelectric field intensities ranging from about 0.1 to 5 kV/cm, preferably0.1 to 1 kV/cm. The nsPEFs can range in time from 1 to 1000 nanoseconds,preferably 1 to 300 nanoseconds. The nsPEFs can also range in electricfield intensity from 1 to 1000 kV/cm, preferably 10 to 350 kV/cm.

In another embodiment, a method of enhancing delivery of drugs to tumorsor other tissues includes applying nanosecond pulse electric fields tosaid tumors or other tissues. The nsPEFs can range in time from 1 to1000 nanoseconds, preferably 1 to 300 nanoseconds. The nsPEFs can alsorange in electric field intensity from 1 to 1000 kV/cm, preferably 10 to350 kV/cm.

In another embodiment, a method of delivering a vaccine to a cellincludes applying nanosecond pulse electric fields to a cell. The nsPEFscan range in time from 1 to 1000 nanoseconds, preferably 1 to 300nanoseconds. The nsPEFs can also range in electric field intensity from1 to 1000 kV/cm, preferably 10 to 350 kV/cm.

In another embodiment, a method of applying nanosecond pulse electricfields is provided in which the nsPEFs are applied to a patient in needof therapy thereof. The nsPEFs can range in time from 1 to 1000nanoseconds, preferably 1 to 300 nanoseconds. The nsPEFs can also rangein electric field intensity from 1 to 1000 kV/cm, preferably 10 to 350kV/cm. The patient in need may, for example, have cancer.

In another embodiment, a method of enhancing gene expression in a cellcomprising applying a nanosecond pulse electric field to said cell isprovided. The nsPEFs can range in time from 1 to 1000 nanoseconds,preferably 1 to 300 nanoseconds. The nsPEFs can also range in electricfield intensity from 1 to 1000 kV/cm, preferably 10 to 350 kV/cm.

In accordance with one or more embodiments of the invention, a pulsegenerator is provided for generating electrical pulses. The pulsegenerator includes a first circuit, a second circuit, and a controlcircuit. The first circuit is used to generate a first pulse having along duration and low voltage amplitude. The second circuit is used togenerate a second pulse having a short duration and high voltageamplitude. The control circuit is provided for controlling the timing ofthe first and second circuits such that the first and second pulses arerespectively generated.

Various embodiments of the invention allow the length of the first pulseto range from 0.001 to 30 milliseconds. The electric field of the firstpulse can also range from 0.1 kV/cm to 5 kV/cm. The length of the secondpulse can vary from 1 to 1000 nanoseconds, while the electric fieldstrength can range from 1 kV/cm to 1000 kV/cm. Furthermore, the controlcircuit can vary the interval between the first and second pulses.

Other embodiments of the invention provide for a first circuit thatincludes a high voltage power supply and a charging resistor coupled tothe high voltage power supply. A capacitor is coupled to the chargingresistor at a first end and coupled to a load at a second end. Atransistor is provided for controlling electrical discharge of thecapacitor to the load. The second circuit can be configured such that itincludes a high voltage power supply, a charging resistor coupled to thehigh voltage power supply; and a transmission line coupled at a firstend to the charging resistor and coupled at a second end to a load. Thetransmission line functions to discharge electricity into the load.

In accordance with at least one embodiment of the invention, a method isprovided for enhancing gene expression using a pulse generator. Themethod comprises the steps: triggering a first pulse having a longduration and low voltage amplitude from a first circuit of the pulsegenerator; delivering the first pulse to at least one cell to causeelectroporation at the plasma membrane of the at least one cell;triggering a second pulse having a long duration and low voltageamplitude from a second circuit of the pulse generator; and deliveringthe second pulse to the at least one cell to cause electroporation atthe nuclear membrane of the at least one cell.

In accordance with one or more embodiments of the invention, a method isprovided for enhancing gene expression in a cell using a multi-pulsegenerator. The method comprises the steps: charging a capacitor;triggering a high voltage, high current transistor to initiate dischargeof the charge accumulated in the capacitor into at least one cell tocause electroporation at the plasma membrane of the at least one cell;triggering the high voltage, high current transistor to stop thedischarge of the capacitor after a predetermined long duration;actuating a switch to decouple the capacitor from the at least one cell;charging a transmission line; triggering a high voltage switch toinitiate discharge of the charge accumulated in the transmission lineinto the at lest one cell to cause electroporation at the nuclearmembrane of the at least one cell; and triggering the high voltageswitch to stop discharge of the transmission line after a predeterminedshort duration.

Other embodiments of the present invention provide for a dual-pulsegenerator for enhancing gene expression in a cell. The dual-pulsegenerator comprises a first pulse generator, a second pulse generator,and a control circuit. The first pulse generator is used to generate afirst pulse having a long duration and low voltage amplitude. The secondpulse generator is used to generate a second pulse having a shortduration and high voltage amplitude. The first pulse causeselectroporation of the cellular plasma membrane of the cell, whilesecond pulse causes electroporation of the nuclear membrane of the cell.The control circuit is used to control the timing of the pulsesgenerated by the first and second pulse generators.

It is to be understood that the invention is not limited in itsapplication to the details of construction and to the arrangements ofthe components set forth in the following description or illustrated inthe drawings. Rather, the invention is capable of other embodiments andof being practiced and carried out in various ways. Also, it is to beunderstood that the phraseology and terminology employed herein are forthe purpose of description and should not be regarded as limiting.

As such, those skilled in the art will appreciate that the conception,upon which this disclosure is based, may readily be utilized as a basisfor the designing of other structures, methods and systems for carryingout the several purposes of the present invention. It is important,therefore, that the claims be regarded as including such equivalentconstructions insofar as they do not depart from the spirit and scope ofthe present invention.

These, and various features of novelty which characterize the invention,are pointed out with particularity in the appended claims forming a partof this disclosure. For a better understanding of the invention, itsoperating advantages and the specific benefits attained by its uses,reference should be had to the accompanying drawings and preferredembodiments of the invention illustrating the best mode contemplated forpracticing the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a typical experiment with HL-60 cells that were exposed, inthe presence of a Green Fluorescent Protein (“GFP”) reporter gene drivenby a constitutive cytomegalovirus (“CMV”) promoter, to a classicalplasma membrane electroporation (long) pulse, a short nsPEF pulse, acombination of a long pulse followed thirty minutes later by a shortnsPEF pulse, or no pulse. The cells shown in FIG. 1 were exposed to along pulse of 3.5 milliseconds (“ms”) and 0.3 kV/cm, a short nsPEF pulseof 10 nanoseconds (“ns”) and 150 kV/cm, or a combination of the long andshort pulse. GFP fluorescence is depicted on the x-axis while the numberof cells fluorescing is depicted on the y-axis. The observed geometricmean GFP fluorescence is listed next to each pulsing condition.

FIG. 2 shows a similar experiment in which HL-60 cells were exposed to along pulse of 450 V and 960 uF, a short nsPEF pulse of 60 ns and 60kV/cm, or a combination of the long and short pulse. The observedgeometric mean GFP fluorescence is listed next to the pulsing conditionsin the inset of the figure.

FIG. 3 shows a similar experiment in which HL-60 cells were exposed to along pulse of 260 V and 960 uF, a short nsPEF pulse of 60 ns and 60kV/cm, or a combination of the long and short pulse. The observedgeometric mean GFP fluorescence is listed next to the pulsing conditionsin the inset of the figure.

FIG. 4 shows a similar experiment in which HL-60 cells were exposed to along pulse of 130 V and 960 uF, a short nsPEF pulse of 60 ns and 60kV/cm, or a combination of the long and short pulse. The observedgeometric mean GFP fluorescence is listed next to the pulsing conditionsin the inset of the figure.

FIG. 5 shows a similar experiment in which HL-60 cells were exposed to along pulse of 450 V and 960 uF, a short nsPEF pulse of 10 ns and 150kV/cm, or a combination of the long and short pulse. The observedgeometric mean GFP fluorescence is listed next to the pulsing conditionsin the inset of the figure.

FIG. 6 shows a similar experiment in which HL-60 cells were exposed to along pulse of 130 V and 960 uF, a short nsPEF pulse of 10 ns and 150kV/cm, or a combination of the long and short pulse. The observedgeometric mean GFP fluorescence is listed next to the pulsing conditionsin the inset of the figure.

FIG. 7 is a circuit diagram for a multi-pulse generator according to oneor more embodiments of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of promoting an understanding of the principles of theinvention, reference will now be made to preferred embodiments andspecific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of theinvention is thereby intended. Alterations and further modifications ofthe invention, and such further applications of the principles of theinvention as illustrated herein, as would normally occur to one skilledin the art to which the invention relates are further contemplatedherein.

For example, features illustrated or described as part of one embodimentcan be used on other embodiments to yield a still further embodiment.Additionally, certain features may be interchanged with similar devicesor features not mentioned yet which perform the same or similarfunctions. It is therefore intended that such modifications andvariations are included within the totality of the present invention.

One aspect of the present invention is directed to a method forintroducing an agent into a cell comprising the application ofnanosecond pulse electric fields (“nsPEF's”) to said cell. As usedherein, the term “agent” includes drugs (e.g., chemotherapeutic agents),nucleic acids (e.g., polynucleotides, genes), peptides and polypeptides(including antibodies), and other molecules for delivery into a cell.“nsPEF's” as used herein are defined as electric pulses in thenanosecond (“ns”) range from 1 to 1000 ns, preferably 1 to 300 ns, withhigh electric field intensities from Ito 1000 kV/cm, preferably 10 to350 kV/cm. The nsPEF conditions defined herein are distinctly differentthan electroporation pulses, not only in their temporal and electricalcharacteristics, but especially in their effects on intact cells andtissues. For comparative purposes, classical electroporation utilizespulses in the microsecond to millisecond range with different pulseshapes (trapezoidal, exponential decaying) and electric fields withstrengths of about 0.1 to 5 kV/cm. The rise times of classicalelectroporation pulses are generally longer than the charging time ofthe cell membrane and, therefore, will not allow an electric field toreach into the cell. By contrast, nsPEF pulses are almost rectangularpulses in the nanosecond range, preferably 10 to 300 ns, with rapid risetimes, short compared to the charging time of the outer cell membraneand ranging from 1 to 30 ns, and high electric fields ranging from about1 to 1000 kV/cm, preferably about 10 to 350 kV/cm. Except for the fastrise and fall times of the pulses, the field strength during the pulseremains at a nearly constant level. In the frequency domain, nsPEFs canbe described as wideband radiation with a cut-off frequency defined bythe inverse of the pulse length, ranging from 1 MHz for a pulse of 1000ns duration to 1 GHz for a 1 ns pulse. But even for lower cut-offfrequencies, the spectra show contributions of higher harmonics,primarily determined by the pulse rise time, up into the GHz range.Furthermore, classical electroporation pulses exhibit energy densitiesin the joules/cc range and power of about 500 W. By contrast, nsPEFpulses exhibit energy densities in the millijoules/cc range, with totalenergies not exceeding 10 J (preferably less than 1 J) and power ofabout 180 MW. About 90% of the energy contained in a nsPEF pulse isapplied in a frequency range up to 60% of the cut-off frequency. Inaddition to the unique short duration and rapid rise time, nsPEFs areexceptional because they are very low energy and extremely high power.Thus, nsPEF pulses can be five to six orders of magnitude shorter, withelectric fields and power several orders of magnitude higher, and energydensities considerably lower than electroporation pulses. Even thoughnsPEF pulses exhibit extremely high power, because their duration is soshort, the energy density does not cause significant thermal effects.

Furthermore, nsPEF pulses and classic electroporation pulses havedramatically different effects on cells. In order to understand thesedifferences, it is necessary to understand the basic effects of anelectric field on a cell. The cell cytoplasm is a conductive body andthe surrounding plasma membrane is a dielectric layer. When cells areplaced in a conductive medium between 2 electrodes and a unipolarvoltage pulse is applied to the electrodes, the resulting current causesaccumulation of electrical charges at the cell membrane and,consequently, a voltage across the membrane. If the membrane voltageexceeds a critical value, structural changes in the surface membraneoccur with trans-membrane pore formation, a process known aselectroporation (Weaver, J. C., Electroporation of cells and tissues,in: J. D. Bronzino (Ed.), The Biomedical Engineering Handbook, CRC andIEEE press, Boca Raton, Fla., 1995, pp. 1431-1440). If the membranevoltage is not excessive and the duration of the pulse is limited,membrane poration can be reversible and the cell survives. The timerequired to charge the surface membrane is dependent upon parameterssuch as the cell diameter (D), resistivities of the cytoplasm (p) andsuspension medium (ρ_(c)), and capacitance of the surface membrane perunit area (eR). For a spherical cell with a surface membrane that is anideal dielectric (no leakage currents), with a diameter of 10 μm,resistivities of cytoplasm and medium of 100 ohm/cm, and a membranecapacitance of 1 μFarad/cm², the charging time constant (τ_(c)) would be75 ns (Cole, K. S. Electric Impedance of Marine Egg Membranes. TransFarady Soc 23:966, 1937) [τ_(c)(ρ_(c)+ρ_(a)/2)cmD/2]. The charging timeconstant is a measure of the time during which the cell interior isexposed to the applied pulsed electric field intensity. A simpleelectrical model for living cells predicts that when the electric pulseduration is reduced into the sub-microsecond range (time domain) thereis an increasing probability that electric field interactions will occurat the level of cell substructures and a decreasing probability that theplasma membrane will be modified. Stated another way, the outer membranebecomes increasingly transparent for oscillating electric fields whenthe angular frequency of the oscillation exceeds a value given by theinverse of the charging time. Therefore, the use of high frequencies andshort durations in the form of nsPEF pulses is more likely to achieveintracellular effects such as the electroporation of intracellularmembranes.

Hence, as the pulse duration decreases, nsPEF pulses bypass the plasmamembrane and target intracellular structures such as the mitochondriaand nucleus, leaving the plasma membrane intact. Therefore, nsPEF pulseshave effects that are different than those of electroporation pulsesbecause, when the pulse duration is short enough and the electric fieldintensity is high enough, intracellular structures are targeted. (Denget al., Biophys. J. 84, 2709-2714 (2003); Beebe et al., IEEE Trans.Plasma Sci. 30:1 Part 2, 286-292 (2002); Beebe et al., FASEB J (2003);Vernier et al., Biochem. Biophys. Res. Comm. 310, 286-295 (2003); Whiteet. al., J Biol. Chem. 279(22):22964-72 (2004); Chen et al., BiochemBiophys Res Commun 317(2):421-7 (2004)). The effects of nsPEF's on cellsdiffer depending on such factors as cell type, pulse duration andrise-time, electric field intensity, and the number of pulses.

In one form of the invention, a desired agent is introduced into a cellusing a known technique (i.e., electroporation, lipid vesicles, viralvectors, co-precipitation with calcium phosphate or dextran). The cellis then exposed to one or more nsPEF pulses in order to facilitatetransfer of the desired agent into the nucleus of the cell. According tothe present invention, the nsPEF pulse can range in duration from 1 to1000 nanoseconds, preferably 1 to 300 nanoseconds. The field amplitudefor the nsPEF pulse can range from 1 to 1000 kV/cm, preferably 10 to 350kV/cm. Experiments on the effects of nsPEFs on the plasma membrane havedemonstrated that nsPEfs cause pores in the plasma membrane to opentransiently, without permanently damaging the cell. (Schoenbach, K. H.,Beebe, S. J., Buescher, E. S., Intracellular effect of ultrashortelectrical pulses, Bioelectromagnetics 22:440-448, 2001). Otherexperiments with nsPEFs have shown that membrane bound organelles in thecell can be opened by the same kind of pulses. (Schoenbach, K. H.,Beebe, S. J., Buescher, E. S., Intracellular effect of ultrashortelectrical pulses, Bioelectromagnetics 22:440-448, 2001). Theoretically,nsPEFs are significantly short enough that the plasma membrane of a cellpulsed with these nsPEFs is not fully charged, thereby avoidingsignificant plasma membrane effects (unlike classical electroporationpulses). Instead, application of nsPEF to a cell results in greatereffects on intracellular membranes. Although not intending to be boundby a particular theory, it is, therefore, hypothesized that nsPEFstemporarily open the nuclear membrane pores without damaging the cell.Therefore, if nsPEF pulses are applied after the desired agent hasalready passed through the plasma membrane of a cell, and the agent isallowed sufficient time to diffuse into the nucleus, nsPEF pulsesfacilitate an increased flux of the agent into the nucleus by opening uppores in the nuclear membrane.

As described earlier, the term “agent” as used herein refers to drugs(e.g., chemotherapeutic agents), nucleic acids (e.g., polynucleotides),and peptides and polypeptides (including antibodies). For example, thepeptide or polypeptide used in the method of the present invention canbe an antigen introduced for the purpose of raising an immune responsein the subject into whose cells it is introduced. Alternatively, thepolypeptide can be a hormone such as calcitonin, parathyroid hormone,erythropoietin, insulin, a cytokine, a lymphokine, a growth hormone, agrowth factor, or a combination of any two or more thereof. Additionalillustrative polypeptides that can be introduced into cells using theinvention method include blood coagulation factors and lymphokines, suchas tumor necrosis factor, interleukins 1, 2 and 3, lymphotoxin,macrophage activating factor, migration inhibition factor, colonystimulating factor, α-interferon, β-interferon, γ-interferon (andsubtypes thereof), and the like.

As used herein, the “nucleic acid,” “nucleic acid molecule,”“polynucleotide”, or “oligonucleotide” of the present invention includesDNA, cDNA, and RNA sequences of all types. For example, thepolynucleotides can be double stranded DNA, single-stranded DNA,complexed DNA, encapsulated DNA, genomic DNA, naked RNA, encapsulatedRNA, a DNA-RNA hybrid, a nucleotide polymer, and combinations thereof.Such agents may be introduced into the cell for any purpose. Forexample, the agents may be used in an amount to modulate cellproliferation or to elicit an immune response, either against thenucleic acid or a protein product encoded by the nucleic acid.

The polynucleotides of the present invention can also be DNA constructs,such as expression vectors. Such expression vectors may encode a desiredgene product (e.g., a gene product homologous or heterologous to thesubject into which it is to be introduced). A therapeutic polypeptide(one encoding a therapeutic gene product) may be operably linked with aregulatory sequence such that the cells of the subject are transfectedwith the therapeutic polypeptide, which is expressed in cells into whichit is introduced according to one aspect of the invention methods. Thepolynucleotide may further encode a selectable marker polypeptide, suchas is known in the art, useful in detecting transformation of cells withagents according to the invention method.

In various embodiments of the invention method, the agent can be a“proliferation-modulating agent,” which alters the proliferativeabilities of cells. Proliferation modulating agents include, but are notlimited to, cytotoxic agents, agents toxic or becoming toxic in thepresence of a protein, and chemotherapeutic agents. The term “cytotoxicagent” refers to a protein, peptide or other molecule having the abilityto inhibit, kill, or lyse a particular cell. Cytotoxic agents includeproteins such as ricin, abrin, diphtheria toxin, saporin, or the like.

In another embodiment, the present invention can be used to facilitatethe enhanced delivery of drugs to tumors and/or other tissues. In thisregard, drugs contemplated for use in the method of the inventioninclude antibiotics such as are known in the art and chemotherapeuticagents having an antitumor or cytotoxic effect. Such drugs or agentsinclude bleomycin, daunomycin, 5-FU, cytosine arabinoside, colchicine,cytochalasin B, daunorubicin, neocarzinostatin, suramin, doxorubicin,carboplatin, taxol, mitomycin C, vincristine, vinblastine, methotrexate,and cisplatin. Other drugs and chemotherapeutic agents will be known tothose of skill in the art (see for example The Merck Index). Such agentscan be “exogenous” agents, which are not normally found in the subject(e.g., chemical compounds) or can also be “endogenous” agents, which arenative to the subject, including suitable naturally occurring agents,such as biological response modifiers (i.e., cytokines, hormones).Additional chemotherapeutic agents include cytotoxic agents derived frommicroorganism or plant sources.

In addition, “membrane-acting” agents can also be introduced into cellsaccording to the invention method. Membrane acting agents are a subsetof chemotherapeutic agents that act primarily by damaging the cellmembrane, such as N-alkylmelamide, and para-chloro mercury benzoate.Alternatively, the composition can include a deoxyribonucleotide analog,such as azidodeoxythymidine, dideoxyinosine, dideoxycytosine,gancyclovir, acyclovir, vidarabine, ribavirin, or any chemotherapeuticknown to those of average skill in the art.

Furthermore, in another embodiment, the methods and apparatus of thepresent invention can be used to administer and enhance the efficacy ofvaccines. Therefore, in an embodiment of the present invention, theagent can be a vaccine. Such a vaccine may consist of inactivatedpathogens, recombinant or natural subunits, and live attenuated or liverecombinant microorganisms. This vaccine may also include apolynucleotide or a protein component.

DNA immunization, a method to induce protective immune responses using“naked” DNA, complexed DNA, or encapsulated DNA, is shown in U.S. Pat.No. 5,589,466. DNA immunization entails the direct, in vivoadministration of vector-based DNA or non-vector DNA that encodes theproduction of defined microbial or cellular antigens, for example, andcytokines (e.g., IL and IFN), for example. The de novo production ofthese antigens in the host's own cells results in the elicitation ofantibody and cellular immune responses that provide protection againstchallenge and persist for extended periods in the absence of furtherimmunizations. The unique advantage of this technology is its ability tomimic the effects of live attenuated vaccines without the safety andstability concerns associated with the parenteral administration of liveinfectious agents. Because of these advantages, considerable researchefforts have focused on refining in vivo delivery systems for naked DNAthat result in, for example, maximal antigen production and resultantimmune responses. Such systems also include liposomes and otherencapsulated means for delivery of DNA.

Therefore, according to the present invention, a DNA or RNA molecule maybe introduced as a vaccine to induce a protective immune response. Inaddition to encoding the gene product (i.e., active agent) to beexpressed, the molecule may also contain initiation and terminationsignals that are operably linked to regulatory elements including apromoter and polyadenylation signal capable of directing expression inthe cells of the vaccinated subject. The vaccine polynucleotide canoptionally be included in a pharmaceutically acceptable carrier asdescribed herein.

As used herein, the term “gene product” refers to a protein or peptideresulting from expression of a polynucleotide within the treated cell.The gene product can be, for example, an immunogenic protein or peptidethat shares at least an epitope with a protein from the pathogen orundesirable cell-type, such as a cancer cell or cells involved inautoimmune disease against which immunization is required. Such proteinsand peptides are antigens and share epitopes with eitherpathogen-associated proteins, proteins associated withhyperproliferating cells, or proteins associated with autoimmunedisorders, depending upon the type of genetic vaccine employed. Theimmune response directed against the antigenic epitope will protect thesubject against the specific infection or disease with which theantigenic epitope is associated. For example, a polynucleotide thatencodes a pathogen-associated gene product can be used to elicit animmune response that will protect the subject from infection by thepathogen.

Likewise, a polynucleotide that encodes a gene product containing anantigenic epitope associated with a hyperproliferative disease such as,for example, a tumor-associated protein, can be used to elicit an immuneresponse directed at hyperproliferating cells. A polynucleotide thatencodes a gene product that is associated with T cell receptors orantibodies involved in autoimmune diseases can be used to elicit animmune response that will combat the autoimmune disease by eliminatingcells in which the natural form of target protein is being produced.Antigenic gene products introduced into cells as active agents accordingto the present invention may be either pathogen-associated proteins,proteins associated with hyperproliferating cells, proteins associatedwith auto-immune disorders or any other protein or peptide known tothose of average skill in the art.

Therefore, in one form of the invention, a desired vaccine is firstintroduced into a cell using known techniques. For example, the vaccinecan first be introduced into the cell and then exposed to one or morensPEF pulses in order to facilitate entry of the vaccine molecules intothe nucleus, thereby stimulating secretion of the antigen produced bythe vaccine molecule.

In addition, it may be desirable to introduce into cells of a subject apolynucleotide that modulates the expression of a gene, such as anendogenous gene, in cells. The term “modulate” envisions the suppressionor augmentation of expression of a gene. Where a cell proliferativedisorder is associated with the expression of a gene, nucleic acidsequences that interfere with the gene's expression at the translationallevel can be used to modulate gene expression. This approach introducesinto the cells of a subject active agents capable of interfering withexpression, such as antisense nucleic acid sequences, ribozymes, ortriplex agents to block transcription or translation of a specific mRNA,either by masking that mRNA with an antisense nucleic acid or triplexagent, or by cleaving it with a ribozyme.

Antisense nucleic acid sequences are DNA or RNA molecules that arecomplementary to at least a portion of a specific mRNA molecule. In thecell, the antisense nucleic acid hybridizes to the corresponding mRNA,forming a double-stranded molecule. The antisense nucleic acidinterferes with the translation of the mRNA, since the cell will nottranslate a mRNA that is double-stranded. Antisense oligomers of about15 nucleotides are preferred, since they are easily synthesized and areless likely than larger molecules to cause problems when introduced intothe target cell. The use of antisense methods to inhibit the in vitrotranslation of genes is well known in the art.

Use of a short oligonucleotide sequence (i.e., “triplex agent”) to stalltranscription is known as the triplex strategy, since the oligomer windsaround double-helical DNA, forming a three-strand helix. Therefore, suchtriplex agents can be designed to recognize a unique site on a chosengene (Maher, et al., Antisense Res. and Dev., 1(3):227, 1991; Helene,C., Anticancer Drug Design, 6(6):569, 1991).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated. There are two basic types ofribozymes namely, tetrahymena-type (Hasselhoff, Nature, 334:585, 1988)and “hammerhead”-type. Tetrahymena-type ribozymes recognize sequencesthat are four bases in length, while “hammerhead”-type ribozymesrecognize base sequences that are 11-18 bases in length. The longer therecognition sequence, the greater the likelihood that the sequence willoccur exclusively in the target mRNA species. Consequently, it ispreferred to employ hammerhead-type ribozymes over tetrahymena-typeribozymes for inactivating a specific mRNA species, and 18-basedrecognition sequences are preferable to shorter recognition sequences asactive agents in one aspect of the invention.

The agent introduced according to the invention methods can also be atherapeutic peptide or polypeptide. For example, immunomodulatory agentsand other biological response modifiers can be administered forincorporation by cells. The term “biological response modifiers” ismeant to encompass substances which are involved in modifying the immuneresponse. Examples of immune response modifiers include such compoundsas lymphokines. Lymphokines include tumor necrosis factor, interleukins1, 2, and 3, lymphotoxin, macrophage activating factor, migrationinhibition factor, colony stimulating factor, alpha-interferon,beta-interferon, and gamma-interferon, their subtypes and the like.

Also included are polynucleotides which encode metabolic enzymes andproteins, including antiangiogenesis compounds, e.g., Factor VIII orFactor IX. The agent of the invention can also be an antibody. The term“antibody” as used herein is meant to include intact molecules as wellas fragments thereof, such as Fab and F(ab′)₂.

The present invention also provides gene therapy for the treatment ofcell proliferative or immunologic disorders mediated by a particulargene or absence thereof. Such therapy would achieve its therapeuticeffect by introduction of a specific sense or antisense polynucleotideinto cells having the disorder. Introduction of polynucleotides into acell can be achieved using a recombinant expression vector such as achimeric virus, or the polynucleotide can be delivered as “naked” DNAfor example. “Introducing” the polynucleotides into a cell encompassesany method of inserting an exogenous nucleic acid molecule into a celland includes, but is not limited to, transduction, transfection,microinjection, and viral infection of the targeted cells.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). When the subject is a human, a vector such as thegibbon ape leukemia virus (GaLV) can be utilized. A number of additionalretroviral vectors can incorporate multiple genes. All of these vectorscan transfer or incorporate a gene for a selectable marker so thattransduced cells can be identified and generated.

In another aspect of the present invention, the polynucleotides can beintroduced into the cell by calcium phosphate and dextranco-precipitation, incorporation of the polynucleotides into lipidvesicles for fusion with the plasma membrane, andelectropermeabilization or electroporation using pulsed electric fieldsto form “pores” in the plasma membrane. Ideally, the choice of a genedelivery system will be made by those of skill in the art, keeping inmind the objectives of efficient gene transfer, with an appropriatelevel of gene expression, in a cell-specific manner, and without anyadverse effects.

The agent introduced into a cell can also include a detectable marker,such as a radioactive label or a fluorescent marker. Alternatively, thecomposition can include a photoactive modification, such as Psoralin C2.Further, the composition can include a phosphoramidate linkage, such asbutylamidate, piperazidate, and morpholidate. Alternatively, thecomposition can include a phosphothioate linkage or ribonucleic acid.These linkages decrease the susceptibility of oligonucleotides andpolynucleotides to degradation in vivo.

In another aspect, the agent of the present invention may be apharmaceutical agent or pharmaceutically active agent. The term“pharmaceutical agent” or “pharmaceutically active agent” as used hereinencompasses any substance that will produce a therapeutically beneficialpharmacological response when administered to a subject, including bothhumans and animals. More than one pharmaceutically active substance maybe included, if desired, in a pharmaceutical composition used in themethod of the present invention.

The pharmaceutically active agent can be employed in various forms, suchas molecular complexes or pharmaceutically acceptable salts.Representative examples of such salts are succinate, hydrochloride,hydrobromide, sulfate, phosphate, nitrate, borate, acetate, maleate,tartrate, salicylate, metal salts (e.g., alkali or alkaline earth),ammonium or amine salts (e.g., quaternary ammonium) and the like.Furthermore, derivatives of the active substances such as esters,amides, and ethers which have desirable retention and releasecharacteristics but which are readily hydrolyzed in vivo byphysiological pH or enzymes can also be employed.

As used herein, the term “therapeutically effective amount” or“effective amount” means that the amount of the biologically active orpharmaceutically active substance is of sufficient quantity and activityto induce a desired pharmacological effect. The amount of substance canvary greatly according to the effectiveness of a particular activesubstance, the age, weight, and response of the individual subject aswell as the nature and severity of the subject's condition or symptoms.Accordingly, there is no upper or lower critical limitation upon theamount of the active agent introduced into the cells of the subject, butshould not be so large as to cause excessive adverse side effects to thecell or tissue containing such cell, such as cytotoxicity, or tissuedamage. The amount required for transformation of cells will vary fromcell type to cell type and from tissue to tissue and can readily bedetermined by those of ordinary skill in the art using the teachingsherein. The required quantity to be employed in the practice of theinvention methods can readily be determined by those skilled in the art.

In one embodiment of the invention method, the amount of active agentsuch as a nucleic acid sequence encoding a gene product introduced intothe cells is a “transforming amount.” A transforming amount is an amountof the active agent effective to modify a cell function, such as mitosisor gene expression, or to cause at least some expression of a geneproduct encoded by the nucleic acid sequence. In other embodiments, theagent may be present in an “immunogenic” amount, an “immuno-modulating”amount, or a “therapeutic amount.” An immunogenic amount is an amount ofthe active agent effective to elicit an immune response. Animmuno-modulating amount is an amount of the active agent effective toalter the immune response in some way. A therapeutic amount is an amountof the active agent effective to induce a desired immunological orbiological response in order to treat a particular disorder for example.

Introduction of active agents across the natural barrier layer of skincan be enhanced by encapsulating the active agent in a controlledrelease vehicle or mixed with a lipid. As used herein with respect topreparations or formulations of active agents, the term “controlledrelease” means that the preparation or formulation requires at least anhour to release a major portion of the active substance into thesurrounding medium, for example, about 1-24 hours, or even longer.

Preferred controlled release vehicles that are suitable forelectrotransport are colloidal dispersion systems, which includemacromolecular complexes, nanocapsules, microcapsules, microspheres,beads, and lipid-based systems, including oil-in-water emulsions,micelles, mixed micelles, liposomes, and the like. For example, in oneembodiment, the controlled release vehicle used to contain the activeagent for microinjection is a biodegradable microsphere. Microspheres,wherein a pharmaceutically active agent is encapsulated by a coating ofcoacervates, is called a “microcapsule.”

Liposomes, which may typically bear a cationic charge, are artificialmembrane vesicles useful as delivery vehicles in vitro and in vivo. Ithas been shown that large unilamellar vesicles (LUV), which range insize from about 0.2 to 4.0 μm, can encapsulate a substantial percentageof an aqueous buffer containing large macromolecules, such as DNA.

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations, making them suitable vehicles forencapsulating an active agent intended to undergo electrotransportaccording to the invention methods.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, gangliosides, and the like. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoyl-phos-phatidylcholine.

Preparations suitable for electrotransport may include the agent with a“pharmaceutically acceptable carrier.” Such carriers are known in theart and include sterile aqueous or non-aqueous solutions, suspensionsand emulsions. Examples of non-aqueous solvents include propyleneglycol, polyethylene glycol, vegetable oils such as olive oil, andinjectable organic esters such as ethyl oleate. Aqueous carriers includewater, alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's, fixed oils, and the like. Vehicles suitable for intercellularor intracellular injection may also include fluid and nutrientreplenishers, electrolyte replenishers, such as those based on Ringer'sdextrose, for example. Preservatives and other additives may also bepresent. For example, antimicrobials, antioxidants, chelating agents,and inert gases may also be used.

It will be appreciated by those of skill in the art that the agent canbe introduced into any desired cell or cell type, including eukaryoticand prokaryotic cells. Non-limiting examples include fat cells, bonecells, vascular cells, muscle cells, cartilage cells, adult, fetal andembryonic stem cells, hematopoeitic cells, lung cells, airway cells,liver cells, intestinal cells, skin cells, nerve cells, and bacteriacells. The methods can also be used to introduce an agent into cancercells, including cancers such as carcinomas, including adenocarcinomas,squamous carcinomas, carcinoma of the organs including breast, bladder,colon, head, neck, prostate, etc.; sarcomas including chondrosarcoma,melanosarcoma, etc.; and leukemia and lymphomas including acutelymphomatic leukemia, acute myelogenous leukemia, non-Hodgkin'slymphoma, Burkitt's lymphoma, B-cell lymphomas, T-cell lymphomas, etc.The methods can also be used to introduce an agent into cells in orderto treat autoimmune disorders, cystic fibrosis, inherited disorders ofhost defense, inherited disorders of carbohydrate metabolism, andinherited disorders of lipid metabolism.

In one embodiment, the present invention is directed to a method ofenhancing gene expression in a cell using nsPEFs. “Gene expression” asused herein is defined as the process by which the information encodedin a gene is converted into protein, peptide, or some form of RNA. Inone form of the invention, cells are placed in the presence ofpolynucleotides being introduced into the cells. The polynucleotide isin a form suitable for introduction into the cell, such as plasmid DNA.The cells and polynucleotides are exposed to relatively long pulses inthe millisecond range. These long pulses cause the outer membranes ofthe cells to open, thereby facilitating the transfer of thepolynucleotides into the cell cytoplasm. The cells are then exposed tonsPEF pulses to facilitate transfer of the polynucleotides into thenucleus. For the long pulses, field amplitudes are low, on the order ofhundreds/low thousands of V/cm. According to the present invention,these long pulses can range in duration from 0.001 to 30 milliseconds,preferably 0.1 to 20 milliseconds. The field amplitudes for the longpulses can range from 0.1 to 5 kV/cm, preferably 0.1 to 1 kV/cm. Duringthe application of the long pulses, the free polynucleotides bindreversibly to the plasma membrane and begin their reversible insertioninto the electropermeabilized membranes. The polynucleotides aretranslocated into the cell not only during pulsation, but also for aconsiderable time afterwards (Karl H. Schoenbach, Sunao Katsuki, RobertH. Stark, Stephen Beebe, and Stephen Buescher, “Bioelectrics—NewApplications for Pulsed Power Technology,” IEEE Trans. Plasma Science30, 293 (2002)). According to the present invention, the nsPEF pulsescan range in duration from 1 to 1000 nanoseconds, preferably 1 to 300nanoseconds. The field amplitudes for the nsPEF pulses can range from 1to 1000 kV/cm, preferably 10 to 350 kV/cm. The application of the nsPEFpulses results in enhanced gene expression.

In order for gene expression to occur, the genes need to enter thenucleus. For long pulses, this process seems to be determined bydiffusion through the nuclear membrane. Therefore, any increase in thepore size of the nuclear membrane causes an increase in the transferrate for genes into the nucleus. If these pulses are applied afterelectropermeabilization of the plasma membrane, and the genes areallowed sufficient time to diffuse into the nucleus, nsPEF pulses allowan increased flux of genes into the nucleus by opening the nuclearmembrane. Alternatively, it is possible that the nsPEFs could promotethe expression of genes through other undefined mechanisms such asenhanced transcription efficiency and/or enhanced transcription of RNA,and/or enhanced translation of protein by mechanisms related or not tocalcium mobilization. Regardless of the mechanism, initial studies thatused electropermeabilization to open the outer plasma membrane, followedby nsPEFs, resulted in increased expression of a green fluorescentprotein (“GFP”) reporter gene in HL-60 cells. The methods and results ofthese experiments are described below.

In another embodiment, the nsPEF pulses alone can be used to enhancegene expression. Because nsPEFs may lead to enhanced transcriptionefficiency, and/or enhanced RNA transcription, and/or enhanced proteintranslation, nsPEFs alone may be applied to a cell in order to enhancegene expression in that cell. The gene or genes enhanced by the presentinvention may be native to the cell and need not necessarily betransfected into the cell. In another embodiment, the nsPEF pulses canbe used to enhance gene expression in cells that have already beentransfected with DNA using any commonly known method described aboveincluding lipid transfer, DNA precipitation with calcium phosphate ordextran, and viral vectors. Following transfection, the nsPEF pulsesfurther facilitate the transport of DNA into the nucleus of the cells.Alternatively, nsPEFs may enhance gene expression by activatingtranscription and/or translation machinery.

NsPEF Pulse Generator

The application of high frequency intracellular effects had been limiteddue to the difficulty of generating large intracellular electric fieldson a time scale that is comparable to or even less than the chargingtime of the surface membrane. If it is assumed that electroporation ofintracellular membranes (intracellular electromanipulation, “IEM”)requires potential differences across such membranes on the order of 1V, electric fields on the order of kV/cm will be needed for poration ofintracellular structures with characteristic dimensions of 1 μm. Most ofthe unipolar pulse generators that have been used in bioelectricexperiments produce microsecond to millisecond duration pulses with arise time too slow to generate measurable intracellular effects.However, as described in U.S. Pat. No. 6,326,177, the present inventorshave developed technology for generating high voltage, short durationelectrical pulses that make it possible to produce electric pulses inthe nanosecond range with voltage amplitudes adequate to generateelectric fields near MV/cm in suspensions of cells or within tissues(Mankowski, J., Kristiansen, M. A review of Short Pulse GeneratorTechnology. IEEE Trans Plasma Science 28:102-108, 2000). Because oftheir nanosecond duration, the average energy transferred to thecells/tissues by these pulses is theoretically negligible, resulting inelectrical effects without accompanying thermal effects.

Furthermore, the preferred embodiment of gene delivery described aboveutilizes a pulse generator that can provide both classicalelectroporation pulses (to open the plasma membrane) and nsPEF pulses(to open the nucleus). Therefore, in one embodiment, the presentinvention is directed to a pulse generator that is capable of deliveringtwo different pulse types in succession in the same apparatus. Thispulse generator may also be able to vary the pulse durations, electricfields, intervals between pulses, and order of the pulses. One pulsetype has a duration in the range of a classical electroporation pulse inthe microsecond or the millisecond range (1 microsecond to 20milliseconds). Such a pulse type is defined herein as a long pulse. Thesecond type of pulse has a duration in the nanosecond range (1 to 300nanoseconds), and defined herein as a short pulse. The time between thelong and short pulses in each set can vary between 0.1 second to severalminutes or hours. Either the long or the short pulse can precede theother. The electric field intensity (kV/cm) of the long and/or the shortpulse in the set can vary.

Accordingly, the apparatus of the present invention can deliver dualpulses differing by these magnitudes in a single apparatus. The optimumtime between pulses will be determined by the diffusion of the agentfrom the outer membrane to the nucleus, and is expected to be in the msrange or longer. Determination of the diffusion time is within thecapabilities of a skilled artisan. The dual-pulse generator may deliverpulses variable in amplitude and duration, as well as in time differencebetween delivery of the pulses, in order to optimize the system fortransfer of the agent into various cells or tissues. The delivery couldbe, for example, a cuvette (for cells in suspension) or two or multiplemetal electrodes for tissue treatment. Other methods of delivery, forexample, in vivo deliver of the pulses, are also envisioned by thepresent invention. Another alternative method of deliver is to use oneor more antennas to deliver the pulse instead of or in addition to anelectrode or cuvette. One or more antennas may be used independently orin conjunction with an electrode or cuvette to deliver the pulse. Theantennas can be, for example, a wide-band antenna, which are used tosuperimpose a plurality of asymmetrical, unipolar pulses to create asingle pulse of the desired duration, for example, the ultrashort pulse.

FIG. 7 is a circuit diagram illustrating an arrangement for a pulsegenerator 100 according to one or more embodiments of the presentinvention. The pulse generator 100 of FIG. 7 is designed to delivers aset of multiple pulse types in succession within the same apparatus.According to one or more embodiments of the invention, the pulsegenerator 100 can be configured to deliver two different pulse types.Thus, the pulse generator 100 of such embodiments can be considered adual-pulse generator 100. Other embodiments of the invention can allowthe pulse generator 100 to deliver more than two pulse types, if andwhen necessary. The pulse generator 100 delivers a first pulse typehaving a duration in the microsecond or millisecond range, and having alow voltage, as will be discussed in greater detail below. This isconsidered a long pulse. For example, the first pulse type can have aduration ranging from 1 microsecond to 20 milliseconds. This range canoptionally be increased or decreased by up to thirty percent (30%)depending on the specific application. The first pulse type is generallyin the same range as a classical electroporation pulse. The second pulsetype is considered a short pulse and has a duration that is less thanthe first pulse type. The second pulse also has a higher voltage thanthe first pulse. For example, the first pulse can have a low voltagevalue in the range of 0.1 to 4 kV, while the second pulse can have ahigher voltage value in the range of 10 to 40 kV, although one or moreembodiments of the present invention can have values ranging up to 50kV. For example, the second pulse type can have a duration in thenanosecond range (e.g., 1 to 1000 nanoseconds). Optionally, the lengthof the second pulse type (or pulse) can also be increased or decreasedby, for example, up to thirty percent (30%). A pause (i.e., time betweenthe pulse types) is provided to separate the long and short pulses.According to one or more embodiments of the present invention, the pausebetween each set of pulses can vary between 0.1 second to severalminutes or hours. Either the long or the short pulse can precede theother. Furthermore, any number of either the long or short pulses can beapplied. Additionally, the electric field intensity (kV/cm) of the longand/or the short pulse in the set can vary, as necessary for variousapplications.

According to at least one embodiment of the present invention, the timebetween pulses can be determined by the diffusion of the agent from theouter membrane to the nucleus. Typically, this time interval is expectedto be in the millisecond range or longer, although physical measurementsof the diffusion would provide better guidance in determining the lengthof the pause. The dual-pulse generator 100 can deliver pulses havingvariable amplitude and duration. The time difference between delivery ofthe pulses can also be varied in order to configure the system for genetransfer into different cells or tissues. The pulses can be delivered invarious ways including, for example, a cuvette for cells in suspension,two or more metal electrodes for tissue treatment, etc. Other methods ofdelivery such as, for example, in vivo delivery of the pulses, are alsoenvisioned by the present invention. Another alternative method ofdeliver is to use one or more antennas (not shown) to deliver the pulseinstead of, or in addition to, an electrode or cuvette. The antennas canbe used independently or in conjunction with an electrode or cuvette todeliver the pulse. According to one or more embodiments of the presentinvention, the antennas can be, for example, wide-band antenna, whichare used to superimpose a plurality of asymmetrical, unipolar pulses tocreate a single pulse of the desired duration. This type of antennaarrangement can be used to deliver a short pulse.

Referring to FIG. 7, the long pulse is generated in a first, low voltagecircuit, shown in the upper left corner of the diagram and generallyreferenced by the numeral 110. A capacitor 112, for example with acapacitance on the order of 1 mF, is charged by a charging resistor,114, using a high voltage power supply 116 (HV). Although FIG. 7 shows acapacitance of 1 mF, other embodiments of the invention can utilizecapacitors having a capacitance ranging from 0.1 mF to 10 mF. Variousother capacitance ratings can be used with the capacitor 112 dependingon the specific application. The resistor 114 can have a resistance, forexample, of 10 kOhms to 10 MOhms, depending on the choice of capacitor.Preferably, the resistor 114 is rated at 1 MOhms to 300 kOhms. Thecapacitor 112 is subsequently discharged into the load 118, which isschematically represented by its resistance (R_(L)). The load 118 couldbe, for example, a cuvette filled with cells in suspension, tissuebetween electrodes, or an apparatus that enables in vitro delivery ofthe pulse. The load resistance, R_(L), is generally presumed to be onthe order of Z=10 Ohms, which may range between 5 to 100 Ohms. Thisrepresents a typical value for cells in a growth medium or buffer insuch commercially available cuvettes. However, the load resistance mayvary according to how the biological sample to be treated is presentedand the apparatus used to deliver the load.

The electrical discharge can be controlled by a transistor 120 such as,for example, an Insulated Gate Bipolar Transistor (“IGBT”), or similarcomponent having a low forward voltage rating. Such transistors 120 aregenerally capable of tolerating currents for a relatively long period oftime without thermal damage. Other types of transistors 120 can also beemployed (e.g., MOSFETs) so long as the transistor 120 is able to handlethe voltage and current being discharged from the capacitor 112. Therise and fall times of such transistors 120 can be in the range of 50 to100 ns. The hold-off voltage of the particular transistor 120 moduleshown in the diagram is V=1.7 kV. For a cuvette with electrode gap of 1cm, it is possible to generate electrical fields of E=1.7 kV/cm. Theelectrical field can be higher if the gap distance, d, is reduced(E=V/d). The closing and opening of the transistor 120, which acts as aswitch, is centrally controlled by means of a control system 122 (orcontrol circuit). The control circuit can be, for example, a delaygenerator, microcontroller, microprocessor, computer controlled circuit,etc.

The second pulse type is generated in a second, high voltage, circuitshown in the lower left corner of the diagram and generally indicated bythe numeral 124. The second circuit 124 can be designed, for example, ina Blumlein configuration as shown in FIG. 7. Two transmission lines 126,or two parallel plates, can be used as an energy reservoir, similar to acapacitor. The transmission lines 126 are charged through a chargingresistor 128 to a high voltage, for example, 50 kV, by means of a dcpower supply 130 (HV). The resistor 128 can have a resistance, forexample, of 10 MOhms to 400 MOhms. According to one or more embodimentsof the present invention, the length of the transmission line 126determines the duration of the short pulse. The duration can becalculated as the length of the transmission line 126 divided by thespeed of light in the dielectric of the transmission line 126. Theimpedance of the transmission lines 126 can be, for example, half of theload resistance. For example, in the case of a 10-Ohm load, theimpedance would be 5 Ohms. The dual-line structure of the Blumleinconfiguration enables full delivery of charge to the load. If, forexample, only one cable were used as the transmission line 126, onlyhalf of the voltage from the power source would be applied. Thus, todeliver 50 kV across the load resistance, the transmission line 126would need to be charged to 100 kV, which may cause technicaldifficulties. Using the dual-line transmission lines 126 enables maximumcharge delivery to the load. Any other double line-type transmissionline 126 could be used in place of the Blumlein configuration.

The second circuit 124 includes a closing switch 132 which can be, forexample, a spark gap: a fast-closing switch designed to close inapproximately one nanosecond, and capable of carrying high currents ofI=V/Z. For example, for a voltage of 50 kV and a load resistance of 10Ohm, the current would be 5000 A. The closing switch 132 delivers theshort, high voltage pulse to the same load 118 as the first circuit 110.As previously discussed, such a load 118 can be, for example, a cuvette,tissue, etc. The closing switch 132 is controlled by a trigger unit 134,which in turn is controlled by the control circuit 122. As previouslydiscussed, the control circuit 122 can be, for example, a delaygenerator, microcontroller, microprocessor, computer controlled circuit,etc. Again, any switch that is capable of withstanding the high voltagesof the pulse can be used in place of the closing switch 132.

In order to prevent the high voltage pulse from damaging and/ordestroying the transistor of the first circuit, at least one embodimentof the present invention provides for separation of the two circuits(110, 124) when the long pulse has been applied. This can be done usingmagnetic switches 136 capable of opening within a time in the range of1-50 milliseconds, and capable of holding approximately 50 kV. Thisopening time is the minimum time between the long pulse and the shortpulse. A trigger unit 138 is provided to actuate the magnetic switches136. The trigger unit 138 can be controlled by, for example, the controlcircuit 122. According to one or more embodiments of the presentinvention, any switch that is reliably capable of holding a high voltagecan be used (e.g., vacuum switches) in place of a magnetic switch.Furthermore, any electrical switch capable of withstanding the highvoltage from the pulses can be used in place of mechanical typeswitches.

The system illustrated in FIG. 7 enables generation of two pulses ofvariable duration and amplitude. The time between the long and shortpulses can also be varied. Instead of having one pulse of each kind, thepulse generator of the present invention is also capable of providingmultiple pulses, both long and short pulses, etc. According to at leastone embodiment of the present invention, the pulses can be controlledbased on programming instructions received by the control circuit 122.Other embodiments of the invention can provide different methods ofcontrolling pulse delivery.

The pulse generator 100 can be controlled by a sequence of instructionsfrom the control circuit 122 shown in FIG. 7. The capacitor 112 can beinitially charged to a voltage determined by its capacitance rating. Thevoltage can vary up to 1.7 kV through the high voltage power supply 116.At time t₁, the transistor 120 is triggered to close. During this time,the capacitor 112 is discharged. If the magnetic switch 136 is alsoclosed, current will flow through the load 118 (R_(L)). The long pulsecan be either exponentially decaying or a square wave, depending on thevalue of the capacitor 112. For example, a square wave can be used forhigher capacitance values.

At time t₂, the long pulse is terminated by triggering the transistor120 to stop discharge of the capacitor 112. Therefore, the time interval(t₂-t₁) determines the duration of the long pulse. The amplitude of thepulse can be controlled by various means, including through the chargingcircuit, the voltage source, the capacitor, and the charging resistor.At time t₃, the trigger unit 138 coupled to the magnetic switch 136 isactuated, thus opening the magnetic switch 136. This action decouplesthe first circuit 110 (low voltage) from the second circuit 124 (highvoltage). At time t₄, trigger unit 134 actuates the closing (or sparkgap) switch 132. The closing switch 132 causes the Blumlein transmissionlines 126 to discharge into the load 118. It should be noted that, priorto discharge, the transmission lines 126 are charged by the power supply130 and the charging resistor 128. This process is similar to chargingthe capacitor 112 of the first circuit 110, as described above. Theultra-short close time of the closing switch 132 enables delivery of theshort, high voltage pulse to the load 118.

The cycle of pulse deliveries can be repeated as often as desired.Additionally, the order of pulse discharged can be altered, for example,such that the short pulse is delivered first. This alternating order ofpulse delivery requires only that the magnetic switch 136 initially bein an open position. Nevertheless, as stated previously, the variousembodiments of the invention can allow for any number of pulses to bedelivered in any order using the pulse generator of the presentinvention (i.e., two short, one long; two long, one short; three short,one long; two long, two short; etc).

According to one of many applications, various embodiments of thepresent invention can be applied to methods of introducing an agent intoa cell. The pulse generator 100 of the present invention can be used toprovide fully adjustable pulsing conditions using either, or both, longand short pulse types within a pulse set. Some of these conditionsinclude, for example, the order (i.e. short pulse first or second),duration, electric field intensity, repetition number, and/or timebetween pulse types within the set. One or more embodiments of thepresent invention allow modification of at least some of these factors(or conditions) to enhance the transfection/expression efficiency of thepulse generator 100 based on classical electroporation factors.Conditions for optimal transfection/expression efficiency differ amongcell types, and can be readily determined by those of skill in the fieldof the invention without undue experimentation.

The foregoing detailed description includes many specific details. Theinclusion of such detail is for the purpose of illustration only andshould not be understood to limit the invention. In addition, featuresin one embodiment may be combined with features in other embodiments ofthe invention. Various changes may be made without departing from thescope of the invention as defined in the following claims.

As one example, the system according to the invention can include ageneral-purpose computer, a specially programmed (special-purpose)computer, control circuitry, controller, etc. User can interact with andprovide input to the pulse generator using various systems, e.g., apersonal computer or PDA, and/or remotely using various protocols totransmit date across a network such as the Internet, an intranet, widearea network (WAN), etc. Moreover, the processing can be controlled by asoftware program on one or more computer systems or processors, or couldeven be partially or wholly implemented in hardware.

User interfaces can be developed in connection with an HTML displayformat. Although HTML is utilized in the illustrated examples, it ispossible to utilize alternative technology (e.g., XML) for displayinginformation, obtaining user instructions and for providing userinterfaces. The system used in connection with the invention may rely onthe integration of various components including, as appropriate and/orif desired, hardware and software servers, database engines, and/orother content providers. The configuration may be, preferably,network-based and uses the Internet as a primary interface with the atleast one user.

The system according to one or more embodiments of the invention maystore collected information and/or indexes to information in one or moredatabases. An appropriate database can be maintained on a standardserver, for example, a small Sun™ Sparc™ or other remote location. Anypresently available or future developed computer software languageand/or hardware components can be employed in the various embodiments ofthe present invention. For example, at least some of the functionalitymentioned above could be implemented using Visual Basic, C, C++, C#, orany assembly language appropriate in view of the processor being used.It could also be written in an interpretive environment such as Java andtransported to multiple destinations to various users.

The many features and advantages of the invention are apparent from thedetailed specification, and thus, it is intended by the appended claimsto cover all such features and advantages of the invention, which fallwithin the true spirit and scope of the invention. Further, sincenumerous modifications and variations will readily occur to thoseskilled in the art, it is not desired to limit the invention to theexact construction illustrated and described, and accordingly, allsuitable modifications and equivalence may be resorted to, fallingwithin the scope of the invention.

Reference will now be made to specific examples illustrating the use ofnsPEFs in enhancing gene expression in a cell. It is to be understoodthat the examples are provided to illustrate preferred embodiments andthat no limitation of the scope of the invention is intended thereby.

Example 1

As stated above, experiments were performed in whichelectropermeabilization of cells was followed by exposure of these cellsto nsPEFs. These experiments resulted in increased expression of a greenfluorescent protein (GFP) reporter gene in HL-60 cells. FIGS. 1-6 showthe effects of different pulses on HL-60 cells.

Materials and Methods

HL-60 cells were removed from growth media, washed, and re-suspended inlow conductivity buffer (LCB) containing 0.85 mM K₂HPO₄, 0.3 mM K₂HPO₄(pH 7.2), and 10 mM KCl (conductivity 1.5 mS/cm at 22° C.). Osmolalitywas adjusted to 290 mOsm by the addition of inositol. Cell suspensions(10⁶ cells/ml) were loaded into the BioRad gene Pulser® cuvettes(Bio-Rad Laboratories, Hercules, Calif.) prior to nsPEF pulsing. A cablepulse generator was use to deliver the NsPEF pulses. NsPEFs weredelivered by means of a cable pulse generator to cells suspended in acuvette with parallel plate electrodes separated by 0.1, 0.2, or 0.4 cm.Briefly, the generator consists of 10Ω pulse-forming network (five 50Ωcables in parallel) and a spark gap in atmospheric air as a nanosecondclosing switch. Post/pulse the cell suspension was removed from thepulsing cuvette and assayed.

The HL-60 cells were exposed to various pulses in the presence of 5 μgof pEGFP, a plasmid containing a nucleotide sequence coding for greenfluorescent protein, downstream of the cytomegalovirus (“CMV”) promoter.The following types of pulses were used: a classical plasma membraneelectroporation (long) pulse administered by a BioRad Gene Pulser, ashort nsPEF pulse, a combination of a long pulse followed 30 minuteslater by a short nsPEF pulse, or no pulse. For instance, in theexperiments depicted in FIG. 1, HL-60 cells were exposed to either aclassical plasma membrane electroporation (long) pulse at 3.5 msec, 0.3kV/cm, a nsPEF pulse at 10 nsec, 150 kV/cm, or a combination of bothpulse types with nsPEF applied 30 minutes after the long pulse. Cellswere then washed, resuspended in growth media, and incubated.Twenty-four hours later, 15,000 cells from the experiment were analyzedby flow cytometry for GFP fluorescence. Fluorescence was expressed asgeometric mean fluorescence as indicated in the figure. The numbers nextto the pulsing conditions in FIGS. 1-6 show the geometric mean GFPfluorescence observed. For instance, in the experiment depicted in FIG.1, the control cells had a mean GFP fluorescence of 3.73, the cellsexposed to the short pulse had a mean GFP fluorescence of 3.58, thecells exposed to the long pulse had a mean GFP fluorescence of 9.67, andthe cells exposed to the combination of pulses had a mean GFPfluorescence of 33.58.

Results of Gene Expression Experiments

As seen in FIG. 1, the nsPEF pulse alone had no effect on GFPfluorescence while the classical electroporation pulse alone increasedfluorescence by about 2.6-fold as determined by the geometric meanfluorescence. However, only about a third of the cells expressed GFP. Inthe presence of both pulses in succession, the GFP fluorescence was33.58. This was about 9-fold greater than the control (3.73) and about3.5-fold greater than that observed for the classical electroporationpulse alone (9.67). Furthermore, essentially all of the cells exposed tothe combination of long and short pulses expressed GFP with greaterfluorescence intensity than cells exposed to classical electroporationconditions.

FIGS. 2-6 show the results of similar experiments in which HL-60 cellswere exposed to various combinations of short pulses (of varying timeranges) and long pulses (of varying voltages). Short pulses were at 60and 150 kV/cm and ranged from 10 to 60 nanoseconds. The long pulses werein the 130 to 450 V/cm range and lasted for 3.5 milliseconds. Theexperiments depicted in these figures similarly demonstrated that cellsexposed to the combination of the long and short pulses exhibited anincrease in mean GFP fluorescence. In these experiments, the combinationof pulses increased GFP fluorescence about 3.2-fold aboveelectroporation pulses alone. For conditions that included 60 ns and 60kV/cm as the nsPEF, GFP fluorescence increased about 3.6-fold. As withthe experiment shown in FIG. 1, essentially all of the cells exposed tothe combination of long and short pulses in FIGS. 2 through 6 expressedGFP with greater fluorescence intensity than cells exposed to classicalelectroporation conditions. These results suggest the potential toincrease gene expression by combining classical electroporation pulseswith nsPEF.

The many features and advantages of the invention are apparent from thedetailed specification, and thus, the appended claims are intended tocover all such features and advantages which fall within the true spiritand scope of the invention. Further, since numerous modifications andvariations will become readily apparent to those skilled in the art, theinvention should not be limited to the exact construction and operationillustrated and described. Rather, all suitable modifications andequivalents may be considered as falling within the scope of the claimedinvention.

What is claimed is:
 1. A method for introducing an agent into a cellcomprising: providing a preparation comprising the cell and an agent;applying a first group of electric field pulses to said preparation,wherein each pulse of the first group comprises a nanosecond pulseelectric field; and applying a second group of electric field pulse tosaid preparation, wherein each pulse of the second group comprises along pulse electric field.
 2. The method of claim 1, wherein thenanosecond pulse electric field pulse is 1 nanosecond to 1000nanoseconds.
 3. The method of claim 1, wherein the nanosecond pulseelectric field pulse is 1 nanosecond to 500 nanoseconds.
 4. The methodof claim 1, wherein the nanosecond pulse electric field pulse is 1nanosecond to 300 nanoseconds.
 5. The method of claim 1, wherein thenanosecond pulse electric field pulse is 10 nanoseconds to 60nanoseconds.
 6. The method of claim 1, wherein each pulse of the firstgroup of electric field pulses has an electric field intensity from 1kV/cm to 1000 kV/cm.
 7. The method of claim 1, wherein each pulse of thefirst group of electric field pulses has an electric field intensityfrom 10 kV/cm to 350 kV/cm.
 8. The method of claim 1, wherein each pulseof the first group of electric field pulses has an electric fieldintensity from 10 kV/cm to 250 kV/cm.
 9. The method of claim 1, whereinthe second group of electric field pulses has a duration of 0.1millisecond to 20 milliseconds.
 10. The method of claim 1, wherein thesecond group of electric field pulses has a duration of 0.001millisecond to 30 milliseconds.
 11. The method of claim 1, wherein eachpulse of the second group of electric field pulses has an electric fieldstrength of 0.1 kV/cm to 5 kV/cm.
 12. The method of claim 1, whereineach pulse of the second group of electric field pulses has an electricfield strength of 0.1 kV/cm to 1 kV/cm.
 13. The method of claim 1further comprising: providing a delay between the first group ofelectric field pulses and the second group of electric field pulses. 14.The method of claim 13, wherein the delay is zero minutes to fiveminutes.
 15. The method of claim 13, wherein the delay is zero minutesto fifteen minutes.
 16. The method of claim 1, wherein the agent isselected from the group comprising drugs, nucleic acids, protein,peptides, and polypeptides.
 17. The method of claim 1, wherein the firstgroup of electric field pulses comprises 1 to 100 pulses.
 18. The methodof claim 1, wherein the second group of electric field pulses comprises1 to 100 pulses.